Mycoplasma species are clearly associated with AIDS. The Mollicutes Mycoplasma fermentans is recovered from HIV positive individuals with much higher frequency than from HIV negative individuals. Mycoplasma pirum and Mycoplasma genitalium have also been recovered. It is certainly less clear what role these and perhaps other yet unrecognized Mycoplasma have in the development of HIV disease. There is no convincing evidence indicating that co-infection with a Mycoplasma, as with HTLV-1, accentuates HIV disease progression. It has been theorized that HIV infection helps the mycoplasmas enter the cell and the mycoplasmas in turn help the HIV virus produce cytopathology. We suggest an alternate theory, mycoplasmas help themselves to get into the host cell possibly already weakened, after they oxidatively damage the host membrane. The resultant mycoplasma-HIV-host interaction is characterized by a stimulation of virus production, maturation or release. Little is known about the biology of these mycoplasmas, especially their physiology, biochemistry and growth response to inhibitors of essential metabolic loci or transport. Such inhibitors are an alternative to tetracycline therapy, to which some AIDS-associated mycoplasmas show resistance. The major problems in studying the interrelationships between mycoplasma and HIV, include: the paucity of information concerning the biology and sensitivities of M. fermentans, M. genitalium and M. pirum, and the apparent difficulty in isolating them and detecting their presence in HIV patients. Initially we will determine and confirm the presence of certain enzymes and metabolic pathways in these AIDS-associated mycoplasmas. We will use these data to 1) construct media for their improved recovery from patients, and to 2) find inhibitors of essential metabolic loci whose selective effect causes impaired growth or death of the mycoplasmas. Two of the most promising essential enzymes are: thymidine kinase and hypoxanthine(guanine)phosphoribosyltransferase. Our previous work with M. genitalium and other Mycoplasma spp. suggest that media additions of nutriments associated with critical metabolic loci will improve isolation and growth of the organisms, these 'hot-spots' involve: fructose phosphate(s), glyceraldehyde-3-phosphate, pyruvate-lactate and the flavin-terminated electron transport system. The optima recovery of AIDS-associated mycoplasmas is also confounded both by their sensitivity to toxic levels of free-fatty acids present in standard media, and to toxic levels of highly reactive oxygenated intermediates produced during the organisms' growth. We speculate that these latter reactive intermediates are also toxic to the host cell that is in intimate physical contact with the mycoplasmas. In experimental murine influenza infection, superoxide anion is reported to be the prime toxic molecule (160). We propose to add to isolation and growth media compounds which will neutralize these toxic substances. Concomitantly, with our studies of metabolism, growth and inhibition, we will develop specific and sensitive PCR reagents to detect AIDS-associated mycoplasmas in host cells. To do this, we will first identify, clone and sequence the essential TK (tk) and H(G)PRT (gpt) genes from M. fermentans, M. genitalium and M. pirum. Employing the data, we will synthesize oligonucleotide primers and use them to identify by PCR the presence of these AIDS-associated mycoplasmas in laboratory infected HSB-2 (pre-CD-4+) cells.